RESUMEN
HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography-mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.
Asunto(s)
Neoplasias de la Mama , Resistencia a Antineoplásicos , Complejo I de Transporte de Electrón , Proteómica , Receptor ErbB-2 , Trastuzumab , Humanos , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Femenino , Complejo I de Transporte de Electrón/metabolismo , Proteómica/métodos , Receptor ErbB-2/metabolismo , Línea Celular Tumoral , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/efectos de los fármacos , Proteoma/metabolismo , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéuticoRESUMEN
Dendritic cells (DCs) are professional APCs used for the development of cancer vaccines because of their ability to activate adaptive immune responses. Previously, we designed the DC/Apo-Nec vaccine using human DCs loaded with dying melanoma cells that primed Ag-specific cytotoxic T cells. Here, we evaluate the effect of a standard pro-inflammatory cytokine cocktail (CC) and adjuvants on DC/Apo-Nec maturation and migration. CC addition to the vaccine coculture allowed efficient Ag uptake while attaining strong vaccine maturation with an immunostimulatory profile. The use of CC not only increased CCR7 expression and the vaccine chemokine responsiveness but also upregulated matrix metalloproteinase-9 secretion, which regulated its invasive migration in vitro. Neither IL-6 nor prostaglandin E2 had a negative effect on vaccine preparation. In fact, all CC components were necessary for complete vaccine maturation. Subcutaneously injected DC/Apo-Nec vaccine migrated rapidly to draining LNs in nude mice, accumulating regionally after 48 h. The migrating cells of the CC-matured vaccine augmented in proportion and range of distribution, an effect that increased further with the topical administration of imiquimod cream. The migrating proportion of human DCs was detected in draining LNs for at least 9 days after injection. The addition of CC during DC/Apo-Nec preparation enhanced vaccine performance by improving maturation and response to LN signals and by conferring a motile and invasive vaccine phenotype both in vitro and in vivo. More importantly, the vaccine could be combined with different adjuvants. Therefore, this DC-based vaccine design shows great potential value for clinical translation.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Citocinas/fisiología , Células Dendríticas/inmunología , Ganglios Linfáticos/inmunología , Melanoma/inmunología , Aminoquinolinas/farmacología , Animales , Movimiento Celular , Quimiotaxis , Humanos , Imiquimod , Metaloproteinasa 9 de la Matriz/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Invasividad Neoplásica , Linfocitos T/inmunologíaRESUMEN
CSF470 vaccine is a mixture of four lethally irradiated melanoma cell lines, administered with BCG and GM-CSF, which is currently being tested in a Phase II/III Clinical trial in stage II/III melanoma patients. To prepare vaccine doses, irradiated melanoma cell lines are frozen using dimethyl sulfoxide (Me(2)SO) and stored in liquid nitrogen (liqN(2)). Prior to inoculation, doses must be thawed, washed to remove Me(2)SO and suspended for clinical administration. Avoiding the use of Me(2)SO and storage in liqN(2) would allow future freeze-drying of CSF470 vaccine to facilitate pharmaceutical production and distribution. We worked on the development of an alternative cryopreservation methodology while keeping the vaccine's biological and immunogenic properties. We tested different freezing media containing trehalose suitable to remain as excipients in a freeze-dried product, to cryopreserve melanoma cells either before or after gamma irradiation. Melanoma cells incorporated trehalose after 5 h incubation at 37°C by fluid-phase endocytosis, reaching an intracellular concentration that varied between 70-140 mM depending on the cell line. Optimal freezing conditions were 0.2 M trehalose and 30 mg/ml human serum albumin, at -84°C. Vaccine doses could be frozen in trehalose at -84°C for at least four months keeping their cellular integrity, antigen expression and apoptosis/necrosis profile after gamma-irradiation as compared to Me(2)SO control. Non-irradiated melanoma cell lines also showed comparable proliferative capacity after both cryopreservation procedures. Trehalose-freezing medium allowed us to cryopreserve melanoma cells, either alive or after gamma irradiation, at -84°C avoiding the use of Me(2)SO and liqN(2) storage. These cryopreservation conditions could be suitable for future freeze-drying of CSF470 vaccine.